**1. Introduction**

T2DM is featured with late onset of the pathology, and represents the form of diabetes which is characterized by insulin resistance, high level of blood sugar, decreased insulin production and pancreatic β-cell deficiency [1]. This state is typically accompanied with suppression of the glucose transport in muscle cells, hepatocytes and adipocytes [2]. Moreover, the increase of blood glucose is accompanied with enhanced degradation of lipids, especially triacylglycerols [3]. Various factors such as hyperglycemia and associated glucotoxicity, lipotoxicity, oxidative stress and mitochondrial dysfunction can induce apoptotic death of ß-cells [4]. As a result of β-cell dysfunction, the fasting-related secretion of the hormone glucagon and the levels of glucose in hepatocytes are not suppressed by subsequent food intake [5]. According to the World Health Organization (WHO), T2DM accounts for 90–95% of the overall number of diabetes cases and its complications are

recognized as the important factors causing blindness, kidney failure, heart attacks, stroke and lower limb amputation [1]. The disease is widely distributed in all parts of human population, and occurs in all regions, including rural areas of low- and middle-income countries. China, India, USA, the countries of Middle East and North Africa are in the top of diabetes occurrence rates [6]. The number of diabetes cases is growing steadily. According to the estimations of the International Diabetes Federation, the occurrence of diabetes will increase from approximately 463 million adults (20–79 years) in 2019 to 700 million by 2045 [7].

The onset of T2DM and its early stages are asymptomatic, as well as a period preceding T2DM and characterized with compromised regulation of blood glucose concentration (so-called pre-diabetes) [8]. Because of this, pre-diabetes is asymptomatically changed by T2DM, and this disease is usually diagnosed at relatively advances stages, when therapy is already required. Thus, to date, approximately every second T2DM case (232 million people) remain undiagnosed [7, 8]. Therefore, timely identifying the individuals in the pre-diabetic state, i.e. with the obvious high risk of developing T2DM is critically important, since early interventions can delay or even prevent onset of the full-scale disease manifestation [9].

Glycation, also often referred to as non-enzymatic glycosylation, represents a reversible reaction of amino and guanidino groups in proteins, peptides, and lipids with reducing sugars (aldoses and ketoses) and carbonyl products of their degradation [10]. The process of protein glycation is often referred to as Maillard reaction of proteins, i.e. the knowledge on its chemistry goes back to the works of Louis Camille Maillard in 1912 [11]. In this reaction, the carbonyl groups of reducing sugars interact with amino/guanidino functions of proteins (mainly with lysine, arginine residues and with N-terminal amino acid residues), with lipids and nucleic acids, yielding early glycation products, also known as Amadori [12] and Heyns compounds [13]. These compounds, often referred to as early glycation products, readily undergo rearrangement, cross-linking, oxidative and non-oxidative degradation, forming so-called advanced glycation end products (AGEs), which are known to accompany not only diabetes complications, but also neurodegenerative diseases and aging [14].

Currently, the Maillard reaction of proteins is considered to be the one of the most common pathways in formation of AGEs (**Figure 1**). Thereby, glycoxidation,

**65**

*Individual Glycation Sites as Biomarkers of Type 2 Diabetes Mellitus*

i.e. oxidative degradation of early glycation products [15] represents the main route of AGE formation, at least for such derivatives as CML and pentosidine [16]. Importantly, α-dicarbonyl compounds, such as 3-deoxyglucosone (3-DG), glyoxal (GO), and methylglyoxal (MGO) are the principle intermediates of AGE formation [17]. Besides glycoxidation, these compounds can also appear as intermediates of glucose autoxidation [18], lipid peroxidation [19], polyol pathway [20], and the Namiki pathway [21]. These α-dicarbonyls are highly relevant for diabetes pathology, as increased concentrations of glyoxal, methylglyoxal, and 3-deoxyglucosone have been found in patients with T2DM [22]. Thus, interac-

represents one of the major and well-characterized AGE. Further cross-linking of GO-derived AGEs with another lysyl residue yields glyoxal-derived lysine dimer (GOLD) or arginine residue forming glyoxal derived imidazolium crosslink (GODIC) [23]. The GO-derived modification of arginine - glyoxal-derived hydro-imidazolinone (Glarg) slowly hydrolyzes under physiological conditions

reaction with arginine produced argpyrimidine or methylglyoxal-derived hydroimidazolone (MG-H1) [17]. The methylglyoxal-lysine dimer (MOLD) is one of the major non-enzymatic cross-links in serum proteins detected during metabolic disorders [25]. Interaction of 3-DG with lysyl residues yields such modifications

In human organism, AGEs exert clearly deleterious effects [27], which are manifested by changes in structure and functions of proteins in human blood. AGEs can interact both with individual proteins *(per se)*, and by direct binding to them through the formation of cross-links. Glycation products are often found in extracellular tissue structures, thus, modified proteins impair matrix–matrix and matrix-cell interactions, leading to reduced cell adhesion, migration and cell death [27]. Intracellular proteins are also readily involved in a broad array of modifications and might lose their functionality at least to some extent [28]. The negative

The term "fructosamine", is a common definition for all ketoamine-containing substances, i.e. Amadori and Heyns compounds, formed during protein glycation [29]. Glycated hemoglobin, which has a lifespan of 90–120 days [29], is routinely used for monitoring of glycemic index in blood of individuals with both T1DM and T2DM. Accumulation of the minor hemoglobin isoform HbA1c, also often referred to as glycated hemoglobin, in blood of diabetic patients was first reported in 1968 [30]. The modification of hemoglobin, underlying this isoform, occurs at the N-terminal valine residue of the β-chain and yields a fructosamine adduct (Amadori

To date, more than 300 different analytical methods and well-established assays for quantitative determination of HbA1c are reported. The most of these methods rely on ion-exchange chromatography (IEC), high-performance liquid chromatography (HPLC) [32], boronate affinity chromatography (BAC) [33], colorimetry [34], as well as different biosensors, based on amperometric [35], potentiometric [36], impedometric [37] and optical sensing [38] techniques. It needs to be taken into account, however, that in some cases determination of HbA1c may appear to be biased or even unreliable. Such cases include pregnant women and patients with end-stage renal disease or those suffering from heavy alcohol consumption [29]. The other well-recognized limitations of the method are its compromised performance with the individuals characterized with increased red blood cell turnover (the state accompanying, for example, hemolytic anemia and severe blood loss) and

interference between different hemoglobin isoforms variants [29].




*DOI: http://dx.doi.org/10.5772/intechopen.95532*

tion of GO with amino function yields *N*<sup>ε</sup>

MGO with lysine leads to the formation of *N*<sup>ε</sup>

as pyrraline, pentosidine, imidazolone or CML [26].

effects of AGEs are summarized on the **Figure 2**.

product) with high diagnostic value [31].

to yield acid-labile *N*<sup>δ</sup>

#### **Figure 1.**

*Formation of early- and advanced glycation in human blood plasma.*

#### *Individual Glycation Sites as Biomarkers of Type 2 Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.95532*

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

adults (20–79 years) in 2019 to 700 million by 2045 [7].

recognized as the important factors causing blindness, kidney failure, heart attacks, stroke and lower limb amputation [1]. The disease is widely distributed in all parts of human population, and occurs in all regions, including rural areas of low- and middle-income countries. China, India, USA, the countries of Middle East and North Africa are in the top of diabetes occurrence rates [6]. The number of diabetes cases is growing steadily. According to the estimations of the International Diabetes Federation, the occurrence of diabetes will increase from approximately 463 million

The onset of T2DM and its early stages are asymptomatic, as well as a period

Currently, the Maillard reaction of proteins is considered to be the one of the most common pathways in formation of AGEs (**Figure 1**). Thereby, glycoxidation,

preceding T2DM and characterized with compromised regulation of blood glucose concentration (so-called pre-diabetes) [8]. Because of this, pre-diabetes is asymptomatically changed by T2DM, and this disease is usually diagnosed at relatively advances stages, when therapy is already required. Thus, to date, approximately every second T2DM case (232 million people) remain undiagnosed [7, 8]. Therefore, timely identifying the individuals in the pre-diabetic state, i.e. with the obvious high risk of developing T2DM is critically important, since early interventions can delay or even prevent onset of the full-scale disease manifestation [9]. Glycation, also often referred to as non-enzymatic glycosylation, represents a reversible reaction of amino and guanidino groups in proteins, peptides, and lipids with reducing sugars (aldoses and ketoses) and carbonyl products of their degradation [10]. The process of protein glycation is often referred to as Maillard reaction of proteins, i.e. the knowledge on its chemistry goes back to the works of Louis Camille Maillard in 1912 [11]. In this reaction, the carbonyl groups of reducing sugars interact with amino/guanidino functions of proteins (mainly with lysine, arginine residues and with N-terminal amino acid residues), with lipids and nucleic acids, yielding early glycation products, also known as Amadori [12] and Heyns compounds [13]. These compounds, often referred to as early glycation products, readily undergo rearrangement, cross-linking, oxidative and non-oxidative degradation, forming so-called advanced glycation end products (AGEs), which are known to accompany not only diabetes complications, but also neurodegenerative

**64**

**Figure 1.**

*Formation of early- and advanced glycation in human blood plasma.*

diseases and aging [14].

i.e. oxidative degradation of early glycation products [15] represents the main route of AGE formation, at least for such derivatives as CML and pentosidine [16]. Importantly, α-dicarbonyl compounds, such as 3-deoxyglucosone (3-DG), glyoxal (GO), and methylglyoxal (MGO) are the principle intermediates of AGE formation [17]. Besides glycoxidation, these compounds can also appear as intermediates of glucose autoxidation [18], lipid peroxidation [19], polyol pathway [20], and the Namiki pathway [21]. These α-dicarbonyls are highly relevant for diabetes pathology, as increased concentrations of glyoxal, methylglyoxal, and 3-deoxyglucosone have been found in patients with T2DM [22]. Thus, interaction of GO with amino function yields *N*<sup>ε</sup> -(carboxymethyl)lysine (CML), which represents one of the major and well-characterized AGE. Further cross-linking of GO-derived AGEs with another lysyl residue yields glyoxal-derived lysine dimer (GOLD) or arginine residue forming glyoxal derived imidazolium crosslink (GODIC) [23]. The GO-derived modification of arginine - glyoxal-derived hydro-imidazolinone (Glarg) slowly hydrolyzes under physiological conditions to yield acid-labile *N*<sup>δ</sup> -(carboxymethyl)arginine (CMA) [24]. The interaction of MGO with lysine leads to the formation of *N*<sup>ε</sup> -(carboxyethyl)lysine (CEL), and reaction with arginine produced argpyrimidine or methylglyoxal-derived hydroimidazolone (MG-H1) [17]. The methylglyoxal-lysine dimer (MOLD) is one of the major non-enzymatic cross-links in serum proteins detected during metabolic disorders [25]. Interaction of 3-DG with lysyl residues yields such modifications as pyrraline, pentosidine, imidazolone or CML [26].

In human organism, AGEs exert clearly deleterious effects [27], which are manifested by changes in structure and functions of proteins in human blood. AGEs can interact both with individual proteins *(per se)*, and by direct binding to them through the formation of cross-links. Glycation products are often found in extracellular tissue structures, thus, modified proteins impair matrix–matrix and matrix-cell interactions, leading to reduced cell adhesion, migration and cell death [27]. Intracellular proteins are also readily involved in a broad array of modifications and might lose their functionality at least to some extent [28]. The negative effects of AGEs are summarized on the **Figure 2**.

The term "fructosamine", is a common definition for all ketoamine-containing substances, i.e. Amadori and Heyns compounds, formed during protein glycation [29]. Glycated hemoglobin, which has a lifespan of 90–120 days [29], is routinely used for monitoring of glycemic index in blood of individuals with both T1DM and T2DM. Accumulation of the minor hemoglobin isoform HbA1c, also often referred to as glycated hemoglobin, in blood of diabetic patients was first reported in 1968 [30]. The modification of hemoglobin, underlying this isoform, occurs at the N-terminal valine residue of the β-chain and yields a fructosamine adduct (Amadori product) with high diagnostic value [31].

To date, more than 300 different analytical methods and well-established assays for quantitative determination of HbA1c are reported. The most of these methods rely on ion-exchange chromatography (IEC), high-performance liquid chromatography (HPLC) [32], boronate affinity chromatography (BAC) [33], colorimetry [34], as well as different biosensors, based on amperometric [35], potentiometric [36], impedometric [37] and optical sensing [38] techniques. It needs to be taken into account, however, that in some cases determination of HbA1c may appear to be biased or even unreliable. Such cases include pregnant women and patients with end-stage renal disease or those suffering from heavy alcohol consumption [29]. The other well-recognized limitations of the method are its compromised performance with the individuals characterized with increased red blood cell turnover (the state accompanying, for example, hemolytic anemia and severe blood loss) and interference between different hemoglobin isoforms variants [29].

#### **Figure 2.**

*Pathological role of AGEs in diabetic complications. 3-deoxyglucosone-derived hydroimidazolone 1 (3DG-H1); activator protein-1 (AP-1),* N<sup>δ</sup> *-(carboxyethyl)arginine (CEA), 3-deoxyglucosonederived lysine dimer (DOLD); glyoxal-derived hydroimidazolone 1 (G-H1), nuclear factor k-light-chain-enhancer of activated B cells (NF-kB), signal transducer and activator of transcription (STAT).*

To some extent, these limitations can be addressed by implementation of a highly abundant plasma protein as a glycation biomarker supplementary to HbA1c. Human serum albumin (HSA) –the major plasma globular polypeptide with a molecular weight of approximately 67 kDa and a serum half-life of about 20 days, is recognized as the best candidate for such kind of biomarker since decades [39]. HSA constitutes up to 70% of the total serum protein, being the most abundant polypeptide in blood plasma [40]. Thus, glycated HSA (often also referred to as glycated albumin, GA) can be employed in determination of glycemic status when the conventional marker (HbA1c) is not reliable enough [41].

Application of GA can be advantageous in comparison to HbA1c due to lower reagent cost and the ability to automate GA analysis on many common laboratory instruments [29]. To date, multiple methods we proposed for analysis of GA. These include enzymatic assays [42], IEC-HPLC [43] and two-dimensional liquid chromatography including affinity chromatography and separation on reversed phase [44]. Immunoassays also represent a promising approach for assessment of GA contents. This strategy can be implemented by radioimmunoassay [45], ELISA [46], enzymelinked boronate immunoassays (ELBIA) [47], colorimetry [48] and electrochemical methods [49]. Method characteristics are summarized in the **Table 1**.

Among the strategies of GA analysis, enzymatic method characterized by shorter operational time and easier performance both in manual and automatic mode [54]. This approach relies on exhaustive hydrolysis of GA by albumin specific proteinase with subsequent oxidation of resulted glycated amino acids by ketoamine oxidase to form hydrogen peroxide, which interacts with chromogen. The colored product can be quantified spectrophotometrically at 546/700 nm. The contents of GA are expressed as the percentage of glycated albumin in total

**67**

**#**

1 2 3 4 5 6 5 6 **Table 1.**

*Overview of analytical techniques employed in analysis of GA. Ultraviolet detector, UV.*

Lateral flow

—

immunoassay

Electrochemistry

—

IEC-HPLC

Colorimetry

Fractionation with

polyethylene glycol

Anion exchange

chromatography, boronate

affinity chromatography

Enzymatic assay

—

ELBIA

Affinity chromatography

UV: absorbance at

492 nm

UV: absorbance at

546/700 nm

UV: absorbance at

546/700 nm

UV: excitation

wavelength 285 nm,

emission wavelength

340 nm

Electro-chemical

Control subjects 2–4%. Diabetic patients ≥16%

[52]

[53]

aptasensor

Colori-metric detection

Control subjects 4.59 ± 0.66 mg/mL (2.44–5.55 mg/mL). Diabetic

patients 7.16 ± 2.58 mg/mL (3.17–17.21 mg/mL)

ELISA

Radioimmuno-assay

**Technique**

**Protein isolation**

Precipitation (ice-cold

trichloroacetic acid solution)

Affinity chromatography

UV: absorbance at

450 nm

(1.6–11.6%)

Control and diabetic subjects: 1.1% - 47.8%

Control subjects 13.4% (range 11.7–16.9%). T2DM 17.4% (14.2–

27.0%) in good control and 26.4% (22.6–49.9%) in poor control

Control subjects: 160–222 (μmol/L). Diabetic patients:

424.6 ± 83.6 μmol/L in T1DM and 346.5 ± 61.6 μmol/L in T2DM

Control subjects 20.2 + 1.6% (range 17.2–23.4%). Diabetic patients:

39.6 + 5.4% in T1DM and 39.4 + 5.9% in T2DM

**Detection** Gamma irradiation

Control subjects: 2.0 ± 0.24 nmol/mg, T2DM: 5.3 ± 2.8 nmol/mg

Control subjects 2.4 ± 0.22%. Diabetic patients: 4.5 ± 1.2%

[45]

[50]

**Value of GA**

*Individual Glycation Sites as Biomarkers of Type 2 Diabetes Mellitus*

[47]

[51]

[48]

[43]

*DOI: http://dx.doi.org/10.5772/intechopen.95532*

**Ref**


#### *Individual Glycation Sites as Biomarkers of Type 2 Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.95532*

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

To some extent, these limitations can be addressed by implementation of a highly abundant plasma protein as a glycation biomarker supplementary to HbA1c. Human serum albumin (HSA) –the major plasma globular polypeptide with a molecular weight of approximately 67 kDa and a serum half-life of about 20 days, is recognized as the best candidate for such kind of biomarker since decades [39]. HSA constitutes up to 70% of the total serum protein, being the most abundant polypeptide in blood plasma [40]. Thus, glycated HSA (often also referred to as glycated albumin, GA) can be employed in determination of glycemic status when

*-(carboxyethyl)arginine (CEA), 3-deoxyglucosonederived lysine* 

*Pathological role of AGEs in diabetic complications. 3-deoxyglucosone-derived hydroimidazolone 1* 

*dimer (DOLD); glyoxal-derived hydroimidazolone 1 (G-H1), nuclear factor k-light-chain-enhancer of* 

Application of GA can be advantageous in comparison to HbA1c due to lower reagent cost and the ability to automate GA analysis on many common laboratory instruments [29]. To date, multiple methods we proposed for analysis of GA. These include enzymatic assays [42], IEC-HPLC [43] and two-dimensional liquid chromatography including affinity chromatography and separation on reversed phase [44]. Immunoassays also represent a promising approach for assessment of GA contents. This strategy can be implemented by radioimmunoassay [45], ELISA [46], enzymelinked boronate immunoassays (ELBIA) [47], colorimetry [48] and electrochemical

the conventional marker (HbA1c) is not reliable enough [41].

*activated B cells (NF-kB), signal transducer and activator of transcription (STAT).*

methods [49]. Method characteristics are summarized in the **Table 1**.

Among the strategies of GA analysis, enzymatic method characterized by shorter operational time and easier performance both in manual and automatic mode [54]. This approach relies on exhaustive hydrolysis of GA by albumin specific proteinase with subsequent oxidation of resulted glycated amino acids by ketoamine oxidase to form hydrogen peroxide, which interacts with chromogen. The colored product can be quantified spectrophotometrically at 546/700 nm. The contents of GA are expressed as the percentage of glycated albumin in total

**66**

**Figure 2.**

*(3DG-H1); activator protein-1 (AP-1),* N<sup>δ</sup>

**Table 1.**

*Overview of analytical techniques employed in analysis of GA. Ultraviolet detector, UV.*

albumin [42]. Although this method was proposed more than 15 years ago, it allows easy and fast quantification of GA with good analytical performance (specificity, accuracy, reproducibility) [55–57]. However, enzymatic assays usually require high concentration of HSA in samples that essentially restricts applicability of the method. Recently, an improved lateral flow immunoassay (LFIA) for simultaneous colorimetric determination of the total HSA and GA, which mostly solves this problem [58].

Due to the wide range of polarities and the different structure of AGEs they can be analyzed by a variety of techniques including spectrofluorimetry, enzyme-linked immunosorbent assays (ELISA), HPLC with UV–VIS detection or coupled on-line to mass spectrometry (MS) [59]. The lack of standardized methods and reference materials increases the risk of analytical errors, negatively affecting accuracy and reproducibility of these methods. Because of this the analysis of AGEs is not widely spread in regular clinical practice [60].

Analysis of total plasma contents of Amadori compounds represents another strategy of glycemic control. Determination of total blood fructosamines provides information on glucose control in the time-frames, mostly limited to the previous two weeks [61]. The total plasma fructosamine content was for the first time used as a diabetes marker in 1983 [62]. The corresponding analytical method relied on the reduction of the dye nitroblue tetrazolium (NBT) to formazan. The level of formazan formation is directly proportional to the fructosamine concentration and can be then assessed by spectrophotometry [62]. The method was significantly improved in 1989 by supplementation of incubation mixtures with a non-ionic detergent in combination with uricase. This made it possible to eliminate the influence of uric acid, lipaemia and polylysine and to provide better sensitivity [63, 64]. Although the method has some disadvantages, such as temperature sensitivity, interference with potential inhibitors of response (such as vitamins and bilirubin) and low standardization, today it remains a valuable tool, which is characterized with ease of handling, low coasts and high potential for automation [29].

Due to a high structural diversity of AGEs their adequate analysis represents a challenging task. In the most easy and straightforward way, analysis of AGEs can rely on their spectral properties, which give access to the total AGE fraction. Indeed, generally, AGEs can be divided into two groups: fluorescent (such as pentosidine or glucosepane) and non-fluorescent AGEs (for example, CML and CEL) as shown on **Figure 3**. Therefore, due to the presence of fluorescent AGEs in the protein structure it is possible to assess AGE-specific fluorescence in serum, urine and saliva using the methods of spectrofluorometry [60]. This AGE-specific fluorescence of cross-linked AGEs can be detected at 440 nm after excitation at 370 nm [65]. For example, using this method, Villa et al. have shown a correlation

#### **Figure 3.**

*Fluorescent (glucosepane, pentosidine) and non-fluorescent (CEL, CMA) AGEs; CEL,* N<sup>ε</sup> *-carboxyethyl lysine; CMA,* N<sup>ω</sup> *-carboxymethylarginine.*

**69**

diabetes mellitus.

*Individual Glycation Sites as Biomarkers of Type 2 Diabetes Mellitus*

between *in vitro* glycated BSA, and the levels of circulating and tissue AGE in diabetic rats [66]. Although this method is simple, fast and cheap, there are some serious limitations, such as lack of detection of non-fluorescent AGEs and interference with non-AGE fluorophores [60]. Furthermore, since more than fifteen years a non-invasive method for *in vivo* determination of AGE-specific autofluorescence is established [67]. However, the presence of endogenous fluorescent signals from cutaneous fluorophores (e.g. nicotinamide adenine dinucleotide, NAD) having the same excitation and emission ranges can interfere with the correct measurement of

Unfortunately, such a powerful method as ELISA is also not free from some intrinsic limitations. Thus, as it can rely not only on monoclonal, but also polyclonal primary antibodies [69], ELISA is often featured with insufficient specificity of antibodies. Moreover, it can suffer from such factors, as high background responses due to significant contents of protein glycation adducts [70] and interference with non-glycated modified or non-modified amino acid residues [71] due to heating and alkaline treatment, implemented in the protocol [72]. Enzyme-linked boronateimmunoassay (ELBIA) represents an efficient extension of ELISA, applicable, however, only to analysis of early glycation products. This technique was first established in 1998 as a method based on the interaction of boronic acids and cis-diols of glycated HSA captured by an anti-HSA antibody [47]. A fully automated ELBIA system, giving access to high-throughput, rapid and precise measurements of GA

It is known since decades, that individual AGEs can be used as biomarkers of different pathologies including diabetes itself. Thereby, individual AGE classes present in biological samples can be assessed by instrumental and immunochemical methods, which need to be more specific due to the targeted character of the analysis. These methods include RP-HPLC, coupled on-line to spectrofluorometry [73], mass spectrometry (MS) [74, 75] or tandem mass spectrometry (MS/MS) [22, 76, 77], as well as gas chromatography - mass spectrometry (GC–MS) [78]. Immunochemical methods are mainly ELISA and Western blotting, using antibodies specific for certain AGE structures [60]. Thus, CML and pentosidine significantly increased in patients with renal failure compared to control subjects [79] and

Like any chronic pathology, diabetes can be efficiently recognized by a set of reliable well-established methods according universal criteria [81]. However, its first manifestations are often invisible for patients and recognized, therefore, already after onset of the pathology [81]. Thus, early diagnosis of T2DM and timely start of its therapy would allow deceleration of the disease progress and reduction the probability of life-threatening complications. Therefore, it is very important to develop a panel of biomarkers, giving access to the early and reliable discovery of

Although HbA1c, fasting blood glucose and glucose tolerance test are well established and universally recognized diagnostic criteria of DM [81], this setup is usually unable to recognize the short term excursions of blood glucose concentrations, which are characterized the beginning of pre-diabetes [82]. Therefore, it was proposed that the biomarkers based on disease-related structural changes of individual proteins might be more sensitive and, hence, more diagnostically efficient [82]. Among such changes, post-translational modifications (PTMs) represent the most promising source of diagnostic information [82–84]. Thereby, modified

was also developed, which was an essential extension of the method.

T2DM compared to non-diabetic controls [80].

**2. Identification of individual glycation sites**

*DOI: http://dx.doi.org/10.5772/intechopen.95532*

total fluorescence [68].

#### *Individual Glycation Sites as Biomarkers of Type 2 Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.95532*

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

spread in regular clinical practice [60].

problem [58].

albumin [42]. Although this method was proposed more than 15 years ago, it allows easy and fast quantification of GA with good analytical performance (specificity, accuracy, reproducibility) [55–57]. However, enzymatic assays usually require high concentration of HSA in samples that essentially restricts applicability of the method. Recently, an improved lateral flow immunoassay (LFIA) for simultaneous colorimetric determination of the total HSA and GA, which mostly solves this

Due to the wide range of polarities and the different structure of AGEs they can be analyzed by a variety of techniques including spectrofluorimetry, enzyme-linked immunosorbent assays (ELISA), HPLC with UV–VIS detection or coupled on-line to mass spectrometry (MS) [59]. The lack of standardized methods and reference materials increases the risk of analytical errors, negatively affecting accuracy and reproducibility of these methods. Because of this the analysis of AGEs is not widely

Analysis of total plasma contents of Amadori compounds represents another strategy of glycemic control. Determination of total blood fructosamines provides information on glucose control in the time-frames, mostly limited to the previous two weeks [61]. The total plasma fructosamine content was for the first time used as a diabetes marker in 1983 [62]. The corresponding analytical method relied on the reduction of the dye nitroblue tetrazolium (NBT) to formazan. The level of formazan formation is directly proportional to the fructosamine concentration and can be then assessed by spectrophotometry [62]. The method was significantly improved in 1989 by supplementation of incubation mixtures with a non-ionic detergent in combination with uricase. This made it possible to eliminate the influence of uric acid, lipaemia and polylysine and to provide better sensitivity [63, 64]. Although the method has some disadvantages, such as temperature sensitivity, interference with potential inhibitors of response (such as vitamins and bilirubin) and low standardization, today it remains a valuable tool, which is characterized with ease

Due to a high structural diversity of AGEs their adequate analysis represents a challenging task. In the most easy and straightforward way, analysis of AGEs can rely on their spectral properties, which give access to the total AGE fraction. Indeed, generally, AGEs can be divided into two groups: fluorescent (such as pentosidine or glucosepane) and non-fluorescent AGEs (for example, CML and CEL) as shown on **Figure 3**. Therefore, due to the presence of fluorescent AGEs in the protein structure it is possible to assess AGE-specific fluorescence in serum, urine and saliva using the methods of spectrofluorometry [60]. This AGE-specific fluorescence of cross-linked AGEs can be detected at 440 nm after excitation at 370 nm [65]. For example, using this method, Villa et al. have shown a correlation

of handling, low coasts and high potential for automation [29].

*Fluorescent (glucosepane, pentosidine) and non-fluorescent (CEL, CMA) AGEs; CEL,* N<sup>ε</sup>

*-carboxyethyl lysine;* 

**68**

**Figure 3.**

*CMA,* N<sup>ω</sup>

*-carboxymethylarginine.*

between *in vitro* glycated BSA, and the levels of circulating and tissue AGE in diabetic rats [66]. Although this method is simple, fast and cheap, there are some serious limitations, such as lack of detection of non-fluorescent AGEs and interference with non-AGE fluorophores [60]. Furthermore, since more than fifteen years a non-invasive method for *in vivo* determination of AGE-specific autofluorescence is established [67]. However, the presence of endogenous fluorescent signals from cutaneous fluorophores (e.g. nicotinamide adenine dinucleotide, NAD) having the same excitation and emission ranges can interfere with the correct measurement of total fluorescence [68].

Unfortunately, such a powerful method as ELISA is also not free from some intrinsic limitations. Thus, as it can rely not only on monoclonal, but also polyclonal primary antibodies [69], ELISA is often featured with insufficient specificity of antibodies. Moreover, it can suffer from such factors, as high background responses due to significant contents of protein glycation adducts [70] and interference with non-glycated modified or non-modified amino acid residues [71] due to heating and alkaline treatment, implemented in the protocol [72]. Enzyme-linked boronateimmunoassay (ELBIA) represents an efficient extension of ELISA, applicable, however, only to analysis of early glycation products. This technique was first established in 1998 as a method based on the interaction of boronic acids and cis-diols of glycated HSA captured by an anti-HSA antibody [47]. A fully automated ELBIA system, giving access to high-throughput, rapid and precise measurements of GA was also developed, which was an essential extension of the method.

It is known since decades, that individual AGEs can be used as biomarkers of different pathologies including diabetes itself. Thereby, individual AGE classes present in biological samples can be assessed by instrumental and immunochemical methods, which need to be more specific due to the targeted character of the analysis. These methods include RP-HPLC, coupled on-line to spectrofluorometry [73], mass spectrometry (MS) [74, 75] or tandem mass spectrometry (MS/MS) [22, 76, 77], as well as gas chromatography - mass spectrometry (GC–MS) [78]. Immunochemical methods are mainly ELISA and Western blotting, using antibodies specific for certain AGE structures [60]. Thus, CML and pentosidine significantly increased in patients with renal failure compared to control subjects [79] and T2DM compared to non-diabetic controls [80].
